Mass spectrometric quantitation for metabolites of leflunomide

ABSTRACT

Methods are described for determining the amount of metabolites of leflunomide in a sample. More specifically, mass spectrometric methods are described for detecting and quantifying teriflunomide in a sample.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/314,093, filed Dec. 7, 2011, which claims priority to U.S.Application Ser. No. 61/428,551, filed Dec. 30, 2010, each of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the detection and quantitation of metabolitesof leflunomide. In particular, the invention relates to methods fordetection and quantitation of metabolites of leflunomide by massspectrometry.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

Leflunomide[N-(4-trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide] is adisease modifying antirheumatic drug (DMARD) with immunomodulatoryactivity. It is indicated for the treatment of active rheumatoidarthritis. Upon absorption, leflunomide is rapidly converted to thepharmacologically active metabolite, α-malononitrilamide A77 1726[2-cyano-3-hydroxy-N-(4-trifluoromethylphenyl)-2-butenamide]. A77 1726(also known as teriflunomide) affects de novo pyrimidine synthesis byinhibition of the enzyme dihydroorotate dehydrogenase (DHODH), therebypreferentially causing cell cycle arrest of autoimmune lymphocytes. Asthe conversion of leflunomide to teriflunomide in vivo is essentiallycomplete, most pharmacokinetic studies have been conducted measuringteriflunomide and not leflunomide.

Various methods, including mass spectrometric methods, have beenreported for detecting and/or quantitating A77 1726. See e.g., Chan V.et al., J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 2004 Apr.25; 803(2):331-5 (reporting a method of determining a leflunomidemetabolite A77 1726 using only HPLC); Sobhani K et al., Am. J. Clin.Pathol. 2010 March; 133(3):454-7 (reporting a method of detecting aleflunomide metabolite using only HPLC); Seah M. S. et al., Drug Metab.Lett. 2008 August; 2(3):153-7 (reporting a method of detecting aleflunomide metabolite using LC and tandem MS in negative ion mode);Parekh J M et al., J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.2010 Aug. 15; 878(24):2217-25 (reporting a method of detecting aleflunomide metabolite using LC-MS-MS in negative ion mode); and E. C.Y. Chan et al., Rapid Commun Mass Spectrom. 2009 February; 23(3):384-94(reporting a method of detecting a leflunomide metabolite using ionmobility spectrometry and time-of-flight mass spectrometry).

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for detecting thepresence or amount of teriflunomide in a sample by mass spectrometry.The methods include subjecting the sample to ionization under conditionssuitable to produce one or more ions detectable by mass spectrometry;determining the amount of one or more ions by mass spectrometry; andusing the amount of one or more ions to determine the presence or amountof teriflunomide in the sample.

In some embodiments, mass spectrometry comprises tandem massspectrometry. In these embodiments, the methods include: a) ionizing thesample under conditions suitable to produce a teriflunomide precursorion; b) fragmenting a teriflunomide precursor ion to produce one or morefragment ions; c) determining the amount of one or more ions produced insteps a) and b); and d) using the amount of the one or more ionsdetermined in step c) to determine the presence or amount ofteriflunomide in the sample. In some embodiments, a teriflunomideprecursor ion with a mass to charge ratio (m/z) of 871.1±0.50 isfragmented to produce one or more fragment ions. In some relatedembodiments, one or more of the fragment ions are selected from thegroup consisting of ions with m/z of 780.6±0.50, 841.8±0.50, 940.8±0.50,1002.5±0.50, 1040.2±0.50, 1083.9±0.50, and 1122.2±0.50. In some relatedembodiments, one or more of the fragment ions are selected from thegroup consisting of ions with m/z of 1040.2±0.50 and 1083.9±0.50.

In another embodiment, the invention provides methods for performing acholestyramine drug elimination procedure. In some embodiments, themethods comprise administering cholestyramine to a patient; obtaining aplasma or serum sample from the patient, and detecting the level ofteriflunomide in the plasma or serum sample by mass spectrometry;wherein, teriflunomide levels of 20 ng/mL or less indicate theeffectiveness of the cholestyramine drug elimination procedure.Teriflunomide levels above 20 ng/mL may indicate that furtheradministration of cholestyramine may be necessary. In these embodiments,detecting the level of teriflunomide in the patient plasma or serum maybe conducted by any of the mass spectrometric procedures describedherein.

In embodiments utilizing tandem mass spectrometry, tandem massspectrometry may be conducted by any method known in the art, includingfor example, multiple reaction monitoring, precursor ion scanning, orproduct ion scanning.

The methods described herein may be capable of detecting teriflunomideat levels within the range of 2.5 ng/mL to 5000 ng/mL, inclusive; suchas within the range of 10 ng/mL to 5000 pg/mL.

In some embodiments, the sample is subjected to an extraction column,such as a solid phase extraction (SPE) column, prior to ionization. Insome related embodiments, SPE and mass spectrometry are conducted withon-line processing.

In some embodiments, the sample is subjected to an analytical column,such as a high performance liquid chromatography (HPLC) column, prior toionization. In some related embodiments, HPLC and mass spectrometry areconducted with on-line processing.

In some embodiments, the methods may be used to determine the presenceor amount of teriflunomide in a biological sample; such as plasma orserum. In some related embodiments, a biological sample is processed byone or more steps to generate a processed sample, which may then besubjected to mass spectrometric analysis. In some embodiments, the oneor more processing steps comprise one or more purification steps, suchas protein precipitation, filtration, liquid-liquid extraction, solidphase extraction, liquid chromatography, any immunopurification process,or the like, and any combination thereof.

In certain preferred embodiments of the methods disclosed herein, massspectrometry is performed in positive ion mode. Alternatively, massspectrometry is performed in negative ion mode. Various ionizationsources, including for example atmospheric pressure chemical ionization(APCI) or electrospray ionization (ESI), may be used in embodiments ofthe present invention. In certain embodiments, teriflunomide is measuredusing ESI in positive ion mode.

In preferred embodiments, a separately detectable internal standard isprovided in the sample, the amount of which is also determined in thesample. In these embodiments, all or a portion of both the analyte ofinterest and the internal standard present in the sample is ionized toproduce a plurality of ions detectable in a mass spectrometer, and oneor more ions produced from each are detected by mass spectrometry. Inthese embodiments, the presence or amount of ions generated from theanalyte of interest may be related to the presence of amount of analyteof interest in the sample.

In other embodiments, the amount of the teriflunomide in a sample may bedetermined by comparison to one or more external reference standards.Exemplary external reference standards include blank plasma or serumspiked with teriflunomide or an isotopically labeled variant thereof.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Purification of the sample by various means may allow relativereduction of one or more interfering substances, e.g., one or moresubstances that may or may not interfere with the detection of selectedparent or daughter ions by mass spectrometry. Relative reduction as thisterm is used does not require that any substance, present with theanalyte of interest in the material to be purified, is entirely removedby purification.

As used herein, the term “immunopurification” or “immunopurify” refersto a purification procedure that utilizes antibodies, includingpolyclonal or monoclonal antibodies, to enrich the one or more analytesof interest Immunopurification can be performed using any of theimmunopurification methods well known in the art. Often theimmunopurification procedure utilizes antibodies bound, conjugated orotherwise attached to a solid support, for example a column, well, tube,gel, capsule, particle or the like Immunopurification as used hereinincludes without limitation procedures often referred to in the art asimmunoprecipitation, as well as procedures often referred to in the artas affinity chromatography.

As used herein, the term “immunoparticle” refers to a capsule, bead, gelparticle or the like that has antibodies bound, conjugated or otherwiseattached to its surface (either on and/or in the particle). In certainembodiments utilizing immunopurification, immunoparticles comprisesepharose or agarose beads. In alternative embodiments utilizingimmunopurification, immunoparticles comprise glass, plastic or silicabeads, or silica gel.

As used herein, the term “sample” refers to any sample that may containan analyte of interest. As used herein, the term “body fluid” means anyfluid that can be isolated from the body of an individual. For example,“body fluid” may include blood, plasma, serum, bile, saliva, urine,tears, perspiration, and the like. In some embodiments, the samplecomprises a body fluid sample; preferably plasma or serum.

As used herein, the term “solid phase extraction” or “SPE” refers to aprocess in which a chemical mixture is separated into components as aresult of an affinity of components dissolved or suspended in a solution(i.e., mobile phase) for a solid through or around which the solution ispassed (i.e., solid phase). SPE, as used herein, is distinct fromimmunopurification in that the affinity of components in the mobilephase to the solid phase is the result of a chemical or physicalinteraction, rather than an immunoaffinity. In some instances, as themobile phase passes through or around the solid phase, undesiredcomponents of the mobile phase may be retained by the solid phaseresulting in a purification of the analyte in the mobile phase. In otherinstances, the analyte may be retained by the solid phase, allowingundesired components of the mobile phase to pass through or around thesolid phase. In these instances, a second mobile phase is then used toelute the retained analyte off of the solid phase for further processingor analysis. SPE, including TFLC, may operate via a unitary or mixedmode mechanism. Mixed mode mechanisms utilize ion exchange andhydrophobic retention in the same column; for example, the solid phaseof a mixed-mode SPE column may exhibit strong anion exchange andhydrophobic retention; or may exhibit column exhibit strong cationexchange and hydrophobic retention.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and turbulent flow liquidchromatography (TFLC) (sometimes known as high turbulence liquidchromatography (HTLC) or high throughput liquid chromatography).

As used herein, the term “high performance liquid chromatography” or“HPLC” (sometimes known as “high pressure liquid chromatography”) refersto liquid chromatography in which the degree of separation is increasedby forcing the mobile phase under pressure through a stationary phase,typically a densely packed column.

As used herein, the term “turbulent flow liquid chromatography” or“TFLC” (sometimes known as high turbulence liquid chromatography or highthroughput liquid chromatography) refers to a form of chromatographythat utilizes turbulent flow of the material being assayed through thecolumn packing as the basis for performing the separation. TFLC has beenapplied in the preparation of samples containing two unnamed drugs priorto analysis by mass spectrometry. See, e.g., Zimmer et al., J ChromatogrA 854: 23-35 (1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368,5,795,469, and 5,772,874, which further explain TFLC. Persons ofordinary skill in the art understand “turbulent flow”. When fluid flowsslowly and smoothly, the flow is called “laminar flow”. For example,fluid moving through an HPLC column at low flow rates is laminar. Inlaminar flow the motion of the particles of fluid is orderly withparticles moving generally in straight lines. At faster velocities, theinertia of the water overcomes fluid frictional forces and turbulentflow results. Fluid not in contact with the irregular boundary “outruns”that which is slowed by friction or deflected by an uneven surface. Whena fluid is flowing turbulently, it flows in eddies and whirls (orvortices), with more “drag” than when the flow is laminar. Manyreferences are available for assisting in determining when fluid flow islaminar or turbulent (e.g., Turbulent Flow Analysis: Measurement andPrediction, P. S. Bernard & J. M. Wallace, John Wiley & Sons, Inc.,(2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott,Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 50 μm. As used in this context, the term“about” means ±10%.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns”, which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. As used in this context, the term “about” means ±10%. In apreferred embodiment the analytical column contains particles of about 5μm in diameter.

As used herein, the terms “on-line” and “inline”, for example as used in“on-line automated fashion” or “on-line extraction” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrometric instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. No. 6,204,500,entitled “Mass Spectrometry From Surfaces;” U.S. Pat. No. 6,107,623,entitled “Methods and Apparatus for Tandem Mass Spectrometry;” U.S. Pat.No. 6,268,144, entitled “DNA Diagnostics Based On Mass Spectrometry;”U.S. Pat. No. 6,124,137, entitled “Surface-Enhanced PhotolabileAttachment And Release For Desorption And Detection Of Analytes;” Wrightet al., Prostate Cancer and Prostatic Diseases 1999, 2: 264-76; andMerchant and Weinberger, Electrophoresis 2000, 21: 1164-67.

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photoionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photoionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber. As the droplets get smaller the electrical surface chargedensity increases until such time that the natural repulsion betweenlike charges causes ions as well as neutral molecules to be released.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to the form of mass spectrometry where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. See, e.g., Robb et al., Anal. Chem.2000, 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase. Laser desorption thermal desorption is a technique wherein asample containing the analyte is thermally desorbed into the gas phaseby a laser pulse. The laser hits the back of a specially made 96-wellplate with a metal base. The laser pulse heats the base and the heatcauses the sample to transfer into the gas phase. The gas phase sampleis then drawn into the mass spectrometer.

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring,” is a detection mode for a mass spectrometricinstrument in which a precursor ion and one or more fragment ions areselectively detected.

As used herein, the term “lower limit of quantification”, “lower limitof quantitation” or “LLOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LOQ isidentifiable, discrete and reproducible with a relative standarddeviation (RSD %) of less than 20% and an accuracy of 85% to 115%.

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as three times the RSD ofthe mean at the zero concentration.

As used herein, an “amount” of an analyte in a body fluid sample refersgenerally to an absolute value reflecting the mass of the analytedetectable in volume of sample. However, an amount also contemplates arelative amount in comparison to another analyte amount. For example, anamount of an analyte in a sample can be an amount which is greater thana control or normal level of the analyte normally present in the sample.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plot of the linearity of quantitation of teriflunomide inspiked mimic serum standards. Details are described in Example 4.

FIG. 2 shows a plot showing the correlation of expected and detectedteriflunomide concentrations in forty spiked serum samples. Details aredescribed in Example 7.

FIG. 3 shows a plot showing the correlation of expected and detectedteriflunomide concentrations in spiked heparin plasma samples. Detailsare described in Example 8.

FIG. 4 shows a plot showing the correlation of expected and detectedteriflunomide concentrations in spiked acid-citrate-dextrose samples.Details are described in Example 8.

FIG. 5 shows a plot showing the correlation of expected and detectedteriflunomide concentrations in spiked EDTA plasma samples. Details aredescribed in Example 8.

DETAILED DESCRIPTION OF THE INVENTION

Methods are described for measuring the amount of teriflunomide in asample. More specifically, mass spectrometric methods are described fordetecting and/or quantifying teriflunomide in a biological sample, suchas human plasma or serum. The methods may utilize liquid chromatographyfollowed by tandem mass spectrometry to quantitate teriflunomide in thesample.

Mean steady state plasma concentrations of teriflunomide from patientson daily dosages of 5, 10, or 25 mg of leflunomide may be expected to beabout 8800 ng/mL, 18,000 ng/mL, and 63,000 ng/mL, respectively. Forpatients who have been prescribed leflunomide, quantitation ofteriflunomide in their plasma may provide a means to confirm compliancewith the prescribed dosages, or may provide a means to determine ifadjustments to prescribed dosages may be necessary.

Further, it is recommended that women of childbearing potential whodiscontinue leflunomide therapy undergo a cholestyramine drugelimination procedure. Such procedures include administration ofcholestyramine for a plurality of consecutive or non-consecutive days,followed by verification that plasma levels of teriflunomide are lessthan 20 ng/mL at least 14 days apart. Thus, methods of the presentinvention provide a means of confirming the effectiveness of acholestyramine drug elimination procedure; that is, methods of thepresent invention may be used to confirm that a sample contains lessthan 20 ng/mL of teriflunomide.

Suitable test samples for use in methods of the present inventioninclude any test sample that may contain the analyte of interest. Insome preferred embodiments, a sample is a biological sample; that is, asample obtained from any biological source, such as an animal, a cellculture, an organ culture, etc. In certain preferred embodiments,samples are obtained from a mammalian animal, such as a dog, cat, horse,etc. Particularly preferred mammalian animals are primates, mostpreferably male or female humans. Preferred samples comprise bodilyfluids such as blood, plasma, serum, saliva, cerebrospinal fluid, ortissue samples; preferably plasma and serum. Such samples may beobtained, for example, from a patient; that is, a living person, male orfemale, presenting oneself in a clinical setting for diagnosis,prognosis, or treatment of a disease or condition. In some embodiments,preferred samples may be obtained from female humans of childbearingpotential. In embodiments where the sample comprises a biologicalsample, the methods may be used to determine the amount of leflunomidemetabolite in the sample when the sample was obtained from thebiological source (i.e., the amount of endogenous leflunomide metabolitein the sample).

The present invention also contemplates kits for a teriflunomidequantitation assay. A kit for a teriflunomide quantitation assay mayinclude a kit comprising the compositions provided herein. For example,a kit may include packaging material and measured amounts of anisotopically labeled internal standard, in amounts sufficient for atleast one assay. Typically, the kits will also include instructionsrecorded in a tangible form (e.g., contained on paper or an electronicmedium) for using the packaged reagents for use in a teriflunomidequantitation assay.

Calibration and QC pools for use in embodiments of the present inventionare preferably prepared using a matrix similar to the intended samplematrix, provided that teriflunomide is essentially absent.

Sample Preparation for Mass Spectrometric Analysis

In preparation for mass spectrometric analysis, teriflunomide may beenriched relative to one or more other components in the sample (e.g.protein) by various methods known in the art, including for example,liquid chromatography, filtration, centrifugation, thin layerchromatography (TLC), electrophoresis including capillaryelectrophoresis, affinity separations including immunoaffinityseparations, extraction methods including ethyl acetate or methanolextraction, and the use of chaotropic agents or any combination of theabove or the like.

Protein precipitation is one method of preparing a test sample,especially a biological test sample, such as serum or plasma. Proteinpurification methods are well known in the art. For example, Polson etal., Journal of Chromatography B 2003, 785:263-275, describes proteinprecipitation techniques suitable for use in methods of the presentinvention. Protein precipitation may be used to remove most of theprotein from the sample leaving teriflunomide in the supernatant. Thesamples may be centrifuged to separate the liquid supernatant from theprecipitated proteins; alternatively the samples may be filtered toremove precipitated proteins. The resultant supernatant or filtrate maythen be applied directly to mass spectrometry analysis; or alternativelyto additional purification methods, such as liquid chromatography, andsubsequent mass spectrometry analysis. In certain embodiments, the useof protein precipitation, such as for example, acetonitrile proteinprecipitation, may obviate the need for TFLC or other on-line extractionprior to mass spectrometry or high performance liquid chromatography(HPLC) and mass spectrometry.

Another method of sample purification that may be used prior to massspectrometry is liquid chromatography (LC). Certain methods of liquidchromatography, including high performance liquid chromatography (HPLC),rely on relatively slow, laminar flow technology. Traditional HPLCanalysis relies on column packing in which laminar flow of the samplethrough the column is the basis for separation of the analyte ofinterest from the sample. The skilled artisan will understand thatseparation in such columns is a partition process and may select LC,including HPLC, instruments and columns that are suitable for use withteriflunomide. The chromatographic column typically includes a medium(i.e., a packing material) to facilitate separation of chemical moieties(i.e., fractionation). The medium may include minute particles. Theparticles typically include a bonded surface that interacts with thevarious chemical moieties to facilitate separation of the chemicalmoieties. One suitable bonded surface is a hydrophobic bonded surfacesuch as an alkyl bonded, cyano bonded, or biphenyl bonded surface. Alkylbonded surfaces may include C-4, C-8, C-12, or C-18 bonded alkyl groups.In preferred embodiments, the column is a biphenyl column. Thechromatographic column includes an inlet port for receiving a sample andan outlet port for discharging an effluent that includes thefractionated sample. The sample may be supplied to the inlet portdirectly, or from a SPE column, such as an on-line extraction column ora TFLC column. In some embodiments, an on-line guard cartridge may beused ahead of the HPLC column to remove particulates and phospholipidsin the samples prior to the samples reaching the HPLC column. In someembodiments, guard cartridge may be a biphenyl guard cartridge.

In one embodiment, the sample may be applied to the LC column at theinlet port, eluted with a solvent or solvent mixture, and discharged atthe outlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytypic (i.e.mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one preferred embodiment, HPLC is conducted with a biphenyl columnchromatographic system. In certain preferred embodiments, a biphenylanalytical column (e.g., a Pinnacle DB Biphenyl analytical column fromRestek Inc. (5 μm particle size, 50×2.1 mm), or equivalent) is used. Incertain preferred embodiments, HPLC is performed using HPLC Grade 0.1%aqueous formic acid as solvent A, and 0.1% formic acid in acetonitrileas solvent B.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

In some embodiments, TFLC may be used for purification of teriflunomideprior to mass spectrometry. In such embodiments, samples may beextracted using a TFLC column which captures the analyte. The analyte isthen eluted and transferred on-line to an analytical HPLC column. Forexample, sample extraction may be accomplished with a TFLC extractioncartridge may be accomplished with a large particle size (50 μm) packedcolumn. Sample eluted off of this column is then transferred on-line toan HPLC analytical column for further purification prior to massspectrometry. Because the steps involved in these chromatographyprocedures may be linked in an automated fashion, the requirement foroperator involvement during the purification of the analyte can beminimized. This feature may result in savings of time and costs, andeliminate the opportunity for operator error.

Detection and Quantitation by Mass Spectrometry

In various embodiments, teriflunomide may be ionized by any method knownto the skilled artisan. Mass spectrometry is performed using a massspectrometer, which includes an ion source for ionizing the fractionatedsample and creating charged molecules for further analysis. For example,ionization of the sample may be performed by electron ionization,chemical ionization, electrospray ionization (ESI), photon ionization,atmospheric pressure chemical ionization (APCI), photoionization,atmospheric pressure photoionization (APPI), laser diode thermaldesorption (LDTD), fast atom bombardment (FAB), liquid secondaryionization (LSI), matrix assisted laser desorption ionization (MALDI),field ionization, field desorption, thermospray/plasmaspray ionization,surface enhanced laser desorption ionization (SELDI), inductivelycoupled plasma (ICP) and particle beam ionization. The skilled artisanwill understand that the choice of ionization method may be determinedbased on the analyte to be measured, type of sample, the type ofdetector, the choice of positive versus negative mode, etc.

Teriflunomide may be ionized in positive or negative mode. In someembodiments, teriflunomide is ionized by ESI in positive mode.

In mass spectrometry techniques generally, after the sample has beenionized, the positively or negatively charged ions thereby created maybe analyzed to determine a mass to charge ratio (m/z). Suitableanalyzers for determining m/z include quadrupole analyzers, ion trapsanalyzers, and time-of-flight analyzers. Exemplary ion trap methods aredescribed in Bartolucci, et al., Rapid Commun. Mass Spectrom. 2000,14:967-73.

According to some methods of the present invention, high resolution/highaccuracy mass spectrometry is used for quantitation of teriflunomide.That is, mass spectrometry is conducted with a mass spectrometer capableof exhibiting a resolving power (FWHM) of at least 10,000, with accuracyof about 50 ppm or less for the ions of interest; preferably the massspectrometer exhibits a resolving power (FWHM) of 20,000 or better andaccuracy of about 20 ppm or less; such as a resolving power (FWHM) of25,000 or better and accuracy of about 5 ppm or less; such as aresolving power (FWHM) of 25,000 or better and accuracy of about 3 ppmor less. Three exemplary mass spectrometers capable of exhibiting therequisite level of performance for teriflunomide ions are those whichinclude orbitrap mass analyzers, certain TOF mass analyzers, or Fouriertransform ion cyclotron resonance mass analyzers.

Elements found in biological active molecules, such as carbon, oxygen,and nitrogen, naturally exist in a number of different isotopic forms.For example, most carbon is present as ¹²C, but approximately 1% of allnaturally occurring carbon is present as ¹³C. Thus, some fraction ofnaturally occurring carbon containing molecules will contain at leastone ¹³C atom. Inclusion of naturally occurring elemental isotopes inmolecules gives rise to multiple molecular isotopic forms. Thedifference in masses of molecular isotopic forms is at least 1 atomicmass unit (amu). This is because elemental isotopes differ by at leastone neutron (mass of one neutron ≈1 amu). When molecular isotopic formsare ionized to multiply charged states, the mass distinction between theisotopic forms can become difficult to discern because massspectrometric detection is based on the mass to charge ratio (m/z). Forexample, two isotopic forms differing in mass by 1 amu that are bothionized to a 5+ state will exhibit differences in their m/z of only 0.2(difference of 1 amu/charge state of 5). High resolution/high accuracymass spectrometers are capable of discerning between isotopic forms ofhighly multiply charged ions (such as ions with charges of ±4, ±5, ±6,±7, ±8, ±9, or higher).

Due to naturally occurring elemental isotopes, multiple isotopic formstypically exist for every molecular ion (each of which may give rise toa separately detectable spectrometric peak if analyzed with a sensitiveenough mass spectrometric instrument). The m/z ratios and relativeabundances of multiple isotopic forms collectively comprise an isotopicsignature for a molecular ion. In some embodiments, the m/z and relativeabundances of two or more molecular isotopic forms may be utilized toconfirm the identity of a molecular ion under investigation. In someembodiments, the mass spectrometric peak from one or more isotopic formsis used to quantitate a molecular ion. In some related embodiments, asingle mass spectrometric peak from one isotopic form is used toquantitate a molecular ion. In other related embodiments, a plurality ofisotopic peaks are used to quantitate a molecular ion. In these laterembodiments, the plurality of isotopic peaks may be subject to anyappropriate mathematical treatment. Several mathematical treatments areknown in the art and include, but are not limited to summing the areaunder multiple peaks or averaging the response from multiple peaks.

In mass spectrometry techniques generally, ions may be detected usingseveral detection modes. For example, selected ions may be detected,i.e. using a selective ion monitoring mode (SIM), or alternatively, masstransitions resulting from collision activated dissociation (CAD), e.g.,multiple reaction monitoring (MRM) or selected reaction monitoring(SRM). CAD is often used to generate fragment ions for furtherdetection. In CAD, precursor ions gain energy through collisions with aninert gas, and subsequently fragment by a process referred to as“unimolecular decomposition.” Sufficient energy must be deposited in theprecursor ion so that certain bonds within the ion can be broken due toincreased vibrational energy. Alternatively, neutral loss may bemonitored.

In some embodiments, the mass-to-charge ratio is determined using aquadrupole analyzer. For example, in a “quadrupole” or “quadrupole iontrap” instrument, ions in an oscillating radio frequency fieldexperience a force proportional to the DC potential applied betweenelectrodes, the amplitude of the RF signal, and the mass/charge ratio.The voltage and amplitude may be selected so that only ions having aparticular mass/charge ratio travel the length of the quadrupole, whileall other ions are deflected. Thus, quadrupole instruments may act asboth a “mass filter” and as a “mass detector” for the ions injected intothe instrument.

One may enhance the specificity of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

Alternate modes of operating a tandem mass spectrometric instrumentinclude product ion scanning and precursor ion scanning. For adescription of these modes of operation, see, e.g., E. Michael Thurman,et al., Chromatographic-Mass Spectrometric Food Analysis for TraceDetermination of Pesticide Residues, Chapter 8 (Amadeo R.Fernandez-Alba, ed., Elsevier 2005) (387).

The results of an analyte assay may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, external standards may be run with thesamples, and a standard curve constructed based on ions generated fromthose standards. Using such a standard curve, the relative abundance ofa given ion may be converted into an absolute amount of the originalmolecule. In certain preferred embodiments, an internal standard is usedto generate a standard curve for calculating the quantity ofteriflunomide. Methods of generating and using such standard curves arewell known in the art and one of ordinary skill is capable of selectingan appropriate internal standard. For example, one or more forms of anisotopically labeled molecule with a similar m/z as teriflunomide may beused as internal standards. In some embodiments described herein, anexemplary internal standard is an isotopically labeled diazepam,although numerous other compounds (isotopically labeled or otherwise)may be used. Numerous other methods for relating the amount of an ion tothe amount of the original molecule will be well known to those ofordinary skill in the art.

As used herein, an “isotopic label” produces a mass shift in the labeledmolecule relative to the unlabeled molecule when analyzed by massspectrometric techniques. Examples of suitable labels include deuterium(²H), ¹³C, and ¹⁵N. One or more isotopic labels can be incorporated atone or more positions in the molecule and one or more kinds of isotopiclabels can be used on the same isotopically labeled molecule.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In particularly preferred embodiments, teriflunomide in a sample isdetected and/or quantified using MS/MS as follows. Samples arepreferably subjected to SPE, then subjected to liquid chromatography,preferably HPLC; the flow of liquid solvent from a chromatographiccolumn enters the heated nebulizer interface of an MS/MS analyzer; andthe solvent/analyte mixture is converted to vapor in the heated chargedtubing of the interface. During these processes, the analyte (i.e.,teriflunomide) is analyzed. The ions, e.g. precursor ions, pass throughthe orifice of the instrument and enter the first quadrupole.Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, allowing selection ofions (i.e., selection of “precursor” and “fragment” ions in Q1 and Q3,respectively) based on their mass to charge ratio (m/z). Quadrupole 2(Q2) is the collision cell, where ions are fragmented. The firstquadrupole of the mass spectrometer (Q1) selects for molecules with themass to charge ratios of teriflunomide. Precursor ions with the correctmass/charge ratios are allowed to pass into the collision chamber (Q2),while unwanted ions with any other mass/charge ratio collide with thesides of the quadrupole and are eliminated. Precursor ions entering Q2collide with neutral argon gas molecules and fragment. The fragment ionsgenerated are passed into quadrupole 3 (Q3), where the fragment ions ofteriflunomide are selected while other ions are eliminated.

The methods may involve MS/MS performed in either positive or negativeion mode; preferably positive ion mode. Using standard methods wellknown in the art, one of ordinary skill is capable of identifying one ormore fragment ions of a particular precursor ion of teriflunomide thatmay be used for selection in quadrupole 3 (Q3).

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC-MS methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, may be measured andcorrelated to the amount of the analyte of interest. In certainembodiments, the area under the curves, or amplitude of the peaks, forfragment ion(s) and/or precursor ions are measured to determine theamount of teriflunomide. As described above, the relative abundance of agiven ion may be converted into an absolute amount of the originalanalyte using calibration standard curves based on peaks of one or moreions of an internal or external molecular standard.

The following Examples serve to illustrate the invention. These Examplesare in no way intended to limit the scope of the methods.

EXAMPLES Example 1 Sample Preparation

Calibrator samples were prepared at eight different concentrations ofteriflunomide in drug free serum (obtained from BioRad). The serumstandards were prepared at concentrations of 10 ng/mL, 25 ng/mL, 75ng/mL, 250 ng/mL, 500 ng/mL, 1000 ng/mL, 2500 ng/mL, and 5000 ng/mL.Control samples were prepared at two different concentrations ofteriflunomide in drug free serum (at ˜100 ng/mL and ˜750 ng/mL).

Patient samples (human serum), calibrator samples, controls, and blankswere prepared for analysis by acetonitrile protein precipitation asfollows.

100 μL of each sample was transferred into a 1.5 ml plasic vial. 20 μLof internal standard solution (1000 mg/mL diazepam-d₅ in methanol) and200 μL of acetonitrile were added to each vial, and the resultingmixture vortexed for about 30 seconds. The vortexed samples wereincubated at room temperature for about 15 minutes and centrifuged forabout 20 minutes at about 5000 rpm. For any sample that was stillcloudy, the supernatant was removed and centrifuged again for about anadditional 2 minutes at about 2000 rpm, and the resulting supernatantremoved again.

200 μL of the resulting supernatants from each sample was transferred toautosampler injection vials for LC-MS/MS analysis as described below.

Example 2 Enrichment of Teriflunomide Using Liquid Chromatography

Injection of 2 μL of each sample was performed with a CTC AnalyticsHTS-PAL system using Analyst 1.5.1 or newer software.

The injected samples were first passed through an on-line RestekPinnacle DB Biphenyl guard cartridge (5 μm, 10×2.1 mm) prior tointroduction into a Restek Pinnacle DP Biphenyl analytical column (5 μm,50×2.1 mm). An HPLC gradient was applied to the analytical column, toseparate teriflunomide from other analytes contained in the sample.Mobile phase A was 0.1% formic acid in HPLC grade water and mobile phaseB was 0.1% formic acid in acetonitrile. The HPLC gradient started with90% mobile phase A for 0.5 minutes, ramped to 80% mobile phase A inapproximately 0.5 minutes, ramped again to 70% mobile phase A inapproximately 0.5 minutes, ramped again to 10% mobile phase A inapproximately 0.3 minutes, and finally ramped back to 90% mobile phase Ain approximately 0.2 minutes, for a total assay run time ofapproximately 2 minutes.

The separated samples are then subjected to MS/MS for quantitation ofteriflunomide.

Example 3 Detection and Quantitation of Teriflunomide by Tandem MS

MS/MS was performed using an Applied Biosystems API4000. Liquidsolvent/analyte exiting the analytical column flowed to the TurboSpray(ESI) interface of the MS/MS analyzer. The solvent/analyte mixture wasconverted to vapor in the heated tubing of the interface, and theresulting vapor was ionized by ESI in positive ion mode.

Teriflunomide ions passed to the first quadrupole (Q1), which selectedions with a m/z of 271.3±0.50. Ions entering quadrupole 2 (Q2) collidedwith nitrogen gas (at a collision cell energy of 17-18 V) to generateion fragments, which were passed to quadrupole 3 (Q3) for furtherselection. Similarly, diazepam-d₅ (internal standard) ions with m/z of290.2±0.50 were selected at Q1, with fragments generated in Q2 andfurther selected in Q3. The following mass transitions were monitoredfor teriflunomide and diazepam-d₅.

TABLE 1 Mass Transitions Observed for Teriflunomide and Diazepam-d₅(Positive Polarity) Precursor Ion Analyte (m/z) Product Ions (m/z)Teruflunomide 271.3 ± 0.50 162.1 ± 0.50 (Quantifier) 142.2 ± 0.50(Qualifier) Diazepam-d₅ 290.2 ± 0.50 198.1 ± 0.50

Of the observed teriflunomide transitions, one was were monitored in MRMmode for quantitative analysis (the precursor ion with m/z of 271.3±0.50to the fragment ion with m/z of 162.1±0.50) and one was monitored as aqualifying transition to confirm the identity of the observed ions (theprecursor ion with m/z of 271.3±0.50 to the fragment ion with m/z of142.2±0.50).

The ratio of the signals of the quantifying transition and qualifyingtransition were evaluated for each sample. Observed ion ratios frompatient samples that are within 20% of the average ratios from thecalibrator samples were considered to confirm the source of ions in thepatient samples.

While this second transition was used for qualification of the observedtransitions, it could have been used to supplement the quantitative datacollected by monitoring the first transition. In fact, additionalproduct ions may be selected to replace or augment either of themonitored transitions.

Example 4 Intra-assay and Inter-assay Precision

The two quality control (QC) pools prepared in Example 1 (withteriflunomide concentrations at ˜100 ng/mL and ˜750 ng/mL) were analyzedto determine intra-assay and inter-assay precision and accuracy.

Five aliquots from each of the two QC pools were analyzed in a fiveassays to determine the reproducibility (Overall CV (%)) across assays.The following values were determined:

TABLE 2 Intra-Assay and Inter-Assay Variation Run 1 Run 2 Run 3 Run 4Run 5 (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) Level 1 1 109.00 115.00130.00 112.00 102.00 2 102.00 106.00 113.00 102.00 106.00 3 103.00131.00 127.00 106.00 125.00 4 93.40 103.00 87.50 109.00 107.00 5 121.00120.00 119.00 122.00 119.00 Count 5 5 5 5 5 Average 105.68 115.00 115.30110.20 111.80 In Run SD 10.21 11.25 16.92 7.56 9.73 Level 2 1 708.00722.00 821.00 779.00 807.00 2 755.00 749.00 753.00 761.00 765.00 3793.00 849.00 814.00 844.00 810.00 4 798.00 826.00 815.00 776.00 728.005 779.00 754.00 673.00 807.00 741.00 Count 5 5 5 5 5 Average 766.60780.00 775.20 793.40 770.20 In Run SD 36.76 54.49 63.49 32.81 37.41Summary Level 1 Level 2 Count 25 25 Grand Mean 111.60 777.08 Pooled WRSD 11.57 46.54 Pooled WR CV 10.36% 5.99% Overall SD 11.16 43.54 OverallCV 10.00% 5.60% Sigma Overall 3.00 5.35 Precision >=3.0 sigma? YES YES

Example 5 Assay Reportable Range and Linearity

To establish the linearity of teriflunomide detection in the assay,seven standards at concentrations ranging from 10 ng/mL to 5000 ng/mL(prepared in Example 1) were analyzed in duplicate. Results of theseanalyses are shown in Table 3.

TABLE 3 Linearity Target Values First Reading Second reading Mean(ng/mL) (ng/mL) (ng/mL) (ng/mL) 10 4.40 8.90 6.65 75 89.30 67.30 78.3250 306.00 372.00 339 500 538.00 697.00 617.5 1000 1140.00 1450.00 12952500 2380.00 3480.00 2930 5000 4870.00 6620.00 5745

A quadratic regression from the two runs yielded a R² value of 0.9993. Agraph showing the linearity of the data is shown in FIG. 1. The assaywas shown to be linear up to 5000 ng/mL, giving a reportable range of 10to 5000 ng/mL.

Example 6 Analytical Sensitivity: Limit of Detection (LOD), Lower Limitof Detection (LLD), and Lower Limit of Quantitation (LOQ)

The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined arbitrarily as two timesthe standard deviation (SD) of the mean from the Zero concentration. Theanalytical sensitivity is the ability of an analytical method todifferentiate and quantify an analyte in the presence of other compoundsin the sample. For selectivity, blank (diluent) samples were obtained,tested for interference, and selectivity ensured at the lower limit ofquantification. Diluent (blank) samples was run in 20 replicates eachand the resulting values statistically analyzed to determine the LOD.

The analytical sensitivity of the assay was determined as the lowerlimit of detection (LLD). The LLD and LOQ were determined by testing 20replicates of sample diluent and calculating the Mean+4SD, while LOQ wasmean+10SD. Data from these analyses is presented in Table 4, below. Asseen in Table 4, the statistical LOQ was 2.24 ng/mL, which is lower thanthe concentration of the lowest standard sample tested (at 10 ng/mL).

TABLE 4 Analyses of blank samples for LOD, LLD, and LOQ REPLICATE # PKAREA  1 49400.00  2 50600.00  3 46500.00  4 43600.00  5 49800.00  649700.00  7 45800.00  8 44600.00  9 42800.00 10 43500.00 11 47700.00 1245000.00 13 48500.00 14 47900.00 15 45800.00 16 49200.00 17 47000.00 1849100.00 19 44500.00 20 50300.00 MEAN 47065 STDEV 2429 LOD (STDEV × 4)9718 STDEV × 10 24295 Variable A = MEAN + (STDEV × 10) 49494 Variable B= PK AREA (STD 1) 110000.00 LOQ BASED ON AREA =(A × 5)/B LOQ BASED ONAREA (ng/mL) 2.249748173

Example 7 Assay Accuracy: Comparison of Clinically Defined Samples

A study of the accuracy of teriflunomide quantitation was carried out bytesting forty spiked samples according to the procedures described inExamples 1-3, and the amounts of detected teriflunomide compared to thespiked values. The results of these analyses are shown in Table 5,below.

TABLE 5 Detection of Teriflunomide in Spiked Serum Samples SpikedTeriflunomide Detected Teriflunomide Recovery Sample (ng/mL) (ng/mL) (%)1 5000 5410 108.20 2 5000 5120 102.40 3 500 513 102.60 4 500 497 99.40 5500 561 112.20 6 1000 1070 107.00 7 1000 1010 101.00 8 1000 1010 101.009 1500 1430 95.33 10 1500 1670 111.33 11 1500 1410 94.00 12 250 23796.80 13 250 242 89.60 14 250 224 89.60 15 500 477 96.60 16 500 483111.40 17 500 557 111.40 18 750 651 100.53 19 750 754 98.80 20 750 74198.80 21 178.5 124 77.31 22 178.5 138 87.39 23 178.5 156 87.39 24 357320 89.64 25 357 309 86.55 26 357 341 95.52 27 100 115 115.00 28 100 9898.00 29 100 85.7 85.70 30 100 83 83.00 31 100 117 117.00 32 750 779103.87 33 750 754 100.53 34 750 673 89.73 35 750 807 107.60 36 750 74198.80 37 850 889 104.59 38 850 826 97.18 39 850 818 96.24 40 850 84198.94

The results of the expected and detected values from the forty spikedsamples correlated with an R²=0.9971 and an intercept of −37.4. A graphshowing the correlation of the data is shown in FIG. 2. Additionally,the mean recovery was 98.7%.

Example 8 Comparison of Specimen Types

Correlation studies similar to that described in Example 6 wereperformed on different specimen types (Heparin, Acid-Citrate-Dextrose(or ACD), and EDTA plasmas) to determine the affect of these differentsample types on quantitation of teriflunomide. For heparin plasma, eightspiked standards at concentrations from about 10-5000 ng/mL wereanalyzed. For ACD and EDTA plasmas, six spiked standards atconcentrations from about 10-5000 ng/mL were analyzed. Results of theseanalyses are shown in Tables 6-8, and the correlations plotted in FIGS.3-5.

TABLE 6 Detection of Teriflunomide in Spiked Heparin Plasma SamplesSpiked Teriflunomide Detected Teriflunomide Recovery Sample  (ng/mL)(ng/mL) (%) HEP1 5000 4460 89.20 HEP2 2300 2330 101.30 HEP3 1000 98298.20 HEP4 500 514 102.80 HEP5 250 256 102.40 HEP6 60 56.5 94.17 HEP7 3034.9 116.33 HEP8 10 12.5 125.00

TABLE 7 Detection of Teriflunomide in Spiked ACD Plasma Samples SpikedTeriflunomide Detected Teriflunomide Recovery Sample (ng/mL) (ng/mL) (%)ACD1 5000 4510 90.20 ACD2 2300 2130 92.61 ACD3 1000 851 85.10 ACD4 400352 88.00 ACD5 200 168 84.00 ACD8 10 10.8 108.00

TABLE 8 Detection of Teriflunomide in Spiked EDTA Plasma Samples SpikedTeriflunomide Detected Teriflunomide Recovery Sample (ng/mL) (ng/mL) (%)EDTA1 5000 4530 90.60 EDTA2 2300 2350 102.17 EDTA3 850 734 86.35 EDTA4500 458 91.60 EDTA5 150 138 92.00 EDTA6 10 11 110.00

The observed correlations were acceptable for all sample types.

Example 9 Interference Studies

Hemolysis Interference: The effects of hemolysis in the assay wereevaluated by spiking various levels of hemoglobin into the 175 ng/mLteriflunomide in drug-free serum to mimic various degrees of hemolysis(low, medium, and high). All samples were analyzed in duplicate. Theresults of these analyses are shown in Table 9.

TABLE 9 Hemolysis Interference Studies for Teriflunomide Second ExpectedFirst Reading Reading Average Recovery Sample (ng/mL) (ng/mL) (ng/mL)(ng/mL) (%) Neat 175 175 175 175 Hem (Low) 175 170 171 170.5 97.4 Hem(Mid) 175 184 179 181.5 103.7 Hem (High) 175 142 139 140.5 80.3

The recovery for all hemolytic samples were within the range ofacceptable results (80%-110%).

Lipid Interference: The effects of lipids in the assay were evaluated byspiking various levels of triglycerides into the 175 ng/mL teriflunomidein drug-free serum to mimic various degrees of lipemic samples (low,medium, and high). All samples were analyzed in duplicate. The resultsof these analyses are shown in Table 10.

TABLE 10 Lipid Interference Studies for Teriflunomide Second ExpectedFirst Reading Reading Average Recovery Sample (ng/mL) (ng/mL) (ng/mL)(ng/mL) (%) Neat 175 175 175 175 Trig (Low) 175 160 155 157.5 90.0 Trig(Mid) 175 146 146 146 83.4 Trig (High) 175 125 114.5 119.5 68.3

The recovery for grossly lipemic samples was not acceptable. Therecovery was outside of the range of acceptable results (80%-110%).

Bilirubin Interference: The effects of bilirubin in the assay wereevaluated by spiking various levels bilirubin into the 175 ng/mLteriflunomide in drug-free serum to mimic various degrees of ictericsamples (low, medium, and high). All samples were analyzed in duplicate.The results of these analyses are shown in Table 11.

TABLE 13 Bilirubin Interference Studies for Teriflunomide First ExpectedReading Second Reading Average Recovery Sample (ng/mL) (ng/mL) (ng/mL)(ng/mL) (%) Neat 175 175 175 175 Bilirubin 175 151 145 148 84.6 (Low)Bilirubin 175 154 142 148 84.6 (Mid) Bilirubin 175 128 138 133 76.0(High)

The recovery for grossly icteric samples was not acceptable. Therecovery was outside of the range of acceptable results (80%-110%).

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for determining the amount ofteriflunomide in a sample by mass spectrometry, said method comprising:a. subjecting the sample to ionization in positive ion mode to produceone or more ions detectable by mass spectrometry; b. determining theamount of one or more ions by mass spectrometry; and c. using the amountof the one or more ions determined in step (b) to determine the amountof teriflunomide in the sample.
 2. The method of claim 1, wherein saidone or more ions determined by mass spectrometry comprise ions with massto charge ratios (m/z) of 271.3±0.50 or 162.1±0.50 or both.
 3. Themethod of claim 2, wherein said mass spectrometry is tandem massspectrometry.
 4. The method of claim 3, wherein tandem mass spectrometryis conducted by multiple reaction monitoring, precursor ion scanning, orproduct ion scanning.
 5. The method of claim 3, wherein said tandem massspectrometry comprises fragmenting a precursor ion with a mass to chargeratio (m/z) of 271.3±0.50 into one or more fragment ions comprising ionswith m/z of 162.1±0.50.
 6. The method of claim 5, wherein the amount ofa fragment ion with m/z of 162.1±0.50 is used to determine the amount ofteriflunomide in a sample.
 7. The method of claim 5, further comprisingconfirming the identity of teriflunomide by detecting an ion with m/z of142.2±0.50.
 8. The method of claim 1, wherein ionization is conductedwith an electrospray ionization (ESI) source.
 9. The method of claim 1,wherein the sample comprises a biological sample.
 10. The method ofclaim 1, wherein the sample comprises plasma or serum.
 11. The method ofclaim 1, wherein the sample is subjected to protein precipitation priorto mass spectrometric ionization.
 12. The method of claim 1, wherein thesample is subjected to liquid chromatography prior to ionization. 13.The method of claim 12, wherein said liquid chromatography compriseshigh performance liquid chromatography.
 14. The method of claim 1,wherein the method is capable of detecting teriflunomide at levelswithin the range of about 2.5 ng/mL to about 5000 ng/mL, inclusive. 15.The method of claim 1, wherein the method is capable of detectingteriflunomide at levels within the range of about 10 ng/mL to about 5000ng/mL, inclusive.
 16. A method for performing a cholestyramine drugelimination procedure, the method comprising: a) obtaining a plasma orserum sample from the patient who has been administered cholestyramine,and b) detecting the amount of teriflunomide in the plasma or serumsample by the method of claim
 1. 17. The method of claim 16, wherein ifthe amount of teriflunomide present in the plasma or serum sample isless than or equal to 20 ng/mL, the cholestyramine drug eliminationprocedure was effective.
 18. The method of claim 16, wherein if theamount of teriflunomide present in the plasma or serum sample is lessthan or equal to 20 ng/mL, the method further comprises repeating stepc) to confirm the first result.
 19. The method of claim 18, wherein ifthe results of two teriflunomide determinations indicate thatteriflunomide is present at levels of less than or equal to 20 ng/mL,the cholestyramine drug elimination procedure was effective.
 20. Themethod of claim 16, wherein if the amount of teriflunomide present inthe plasma or serum sample is greater than 20 ng/mL, the method furthercomprises repeating steps a)-b).